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miR-17 promotes expansion and adhesion of human cord blood CD34(+) cells in vitro.

Yang Y, Wang S, Miao Z, Ma W, Zhang Y, Su L, Hu M, Zou J, Yin Y, Luo J - Stem Cell Res Ther (2015)

Bottom Line: However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells.The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical & Research Technology, School of Medicine, University of Maryland, Baltimore, MD, 21201, USA. yangyx@bjmu.edu.cn.

ABSTRACT

Introduction: We have recently found that miR-17 is necessary in the cell-extrinsic control of cord blood (CB) CD34(+) cell function. Here, we demonstrated that the proper level of miR-17 is also necessary in the cell-intrinsic control of the hematopoietic properties of CB CD34(+) cells.

Methods: The miR-17 overexpression and knockdown models were created using primary CB CD34(+) cells transfected by the indicated vectors. Long-term culture, colony forming, adhesion and trans-well migration assays were carried out to investigate the function of miR-17 on CB CD34(+) cells in vitro. NOD prkdc (scid) Il2rg () mice were used in a SCID repopulating cell assay to investigate the function of miR-17 on CB CD34(+) cells in vivo. A two-tailed Student's t-test was used for statistical comparisons.

Results: In vitro assays revealed that ectopic expression of miR-17 promoted long-term expansion, especially in the colony-forming of CB CD34(+) cells and CD34(+)CD38(-) cells. Conversely, downregulation of miR-17 inhibited the expansion of CB CD34(+) cells. However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells. The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.

Conclusion: We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

No MeSH data available.


Related in: MedlinePlus

Effect of miR-17 modulation on the hematopoietic reconstitution potential of CB CD34+ cells in NPG mice. a Effect of miR-17 modulation on repopulation of CB CD34+ cells in NOD prkdcscid Il2rg (NPG™) mice. 4.0 × 104miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) CB CD34+ cells were injected intravenously into the sublethally irradiated NPG mice (n = 6 per group). The PB of NPG recipients was temporally monitored by analyzing CD45+ population every 4 weeks. The mice were sacrificed at 20 weeks after transplantation and the mononuclear cells from bone marrow were analyzed for human cells composed of CD45+, CD45−CD36+ and CD36−GPA+ cells and CD45+CD34+ population by flow cytometry. The level of total human cell engraftment is shown in the left panel. p < 0.03 or 0.04, between the NPG mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells (Student’s t-test). The fraction of CD45+, erythroid (CD45−CD36+ and CD36−GPA+) and CD45+CD34+ population among the engrafted human cells is shown in the middle panel. The percent of CD45+ cells of PB from NPG recipients was shown in the right panel. *p < 0.05, between the NPG mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells (Student’s t-test). The significant difference was analyzed between the mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells. b Flow cytometry analysis of the human CB CD34+ cell repopulation in a representative NPG mouse after miR-17 modulation. Fresh CD34+ cells served as controls. The MNCs from bone marrow harvested from the engrafted NPG mice were examined by flow cytometry for the assessment of human cells composed of CD45+ cells (R1), erythroid cells including CD45−CD36+ (R2) and CD36−GPA+ (R3) populations and CD45+CD34+ cells (R4). c The levels of miR-17 expression in GFP-positive cells from engrafted mice at 20 weeks were tested by real-time PCR. d The bone marrow MNCs containing the different percentage of human cells (lanes 2–5) from the representative engrafted mice were analyzed for human-specific 17α-satellite DNA by PCR. The human-specific 17α-satellite gene was detected when the human cells were over 0.50 % (lanes 3–5) whereas it was indetectable at a percentage of 0.21 % (lane 2). Lane 1, one mouse without transplants; lane 2, one mouse receiving transplants of 17/OE CD34+ cells; lane 3, one mouse receiving transplants of 17/KD CD34+ cells; lane 4, one mouse receiving transplants of CTRL CD34+ cells; lane 5, one mouse receiving transplants of fresh CD34+ cells
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Fig3: Effect of miR-17 modulation on the hematopoietic reconstitution potential of CB CD34+ cells in NPG mice. a Effect of miR-17 modulation on repopulation of CB CD34+ cells in NOD prkdcscid Il2rg (NPG™) mice. 4.0 × 104miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) CB CD34+ cells were injected intravenously into the sublethally irradiated NPG mice (n = 6 per group). The PB of NPG recipients was temporally monitored by analyzing CD45+ population every 4 weeks. The mice were sacrificed at 20 weeks after transplantation and the mononuclear cells from bone marrow were analyzed for human cells composed of CD45+, CD45−CD36+ and CD36−GPA+ cells and CD45+CD34+ population by flow cytometry. The level of total human cell engraftment is shown in the left panel. p < 0.03 or 0.04, between the NPG mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells (Student’s t-test). The fraction of CD45+, erythroid (CD45−CD36+ and CD36−GPA+) and CD45+CD34+ population among the engrafted human cells is shown in the middle panel. The percent of CD45+ cells of PB from NPG recipients was shown in the right panel. *p < 0.05, between the NPG mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells (Student’s t-test). The significant difference was analyzed between the mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells. b Flow cytometry analysis of the human CB CD34+ cell repopulation in a representative NPG mouse after miR-17 modulation. Fresh CD34+ cells served as controls. The MNCs from bone marrow harvested from the engrafted NPG mice were examined by flow cytometry for the assessment of human cells composed of CD45+ cells (R1), erythroid cells including CD45−CD36+ (R2) and CD36−GPA+ (R3) populations and CD45+CD34+ cells (R4). c The levels of miR-17 expression in GFP-positive cells from engrafted mice at 20 weeks were tested by real-time PCR. d The bone marrow MNCs containing the different percentage of human cells (lanes 2–5) from the representative engrafted mice were analyzed for human-specific 17α-satellite DNA by PCR. The human-specific 17α-satellite gene was detected when the human cells were over 0.50 % (lanes 3–5) whereas it was indetectable at a percentage of 0.21 % (lane 2). Lane 1, one mouse without transplants; lane 2, one mouse receiving transplants of 17/OE CD34+ cells; lane 3, one mouse receiving transplants of 17/KD CD34+ cells; lane 4, one mouse receiving transplants of CTRL CD34+ cells; lane 5, one mouse receiving transplants of fresh CD34+ cells

Mentions: To further support our in vitro expansion results, we examined the hematopoietic reconstitution potential of CB CD34+ cells after miR-17 modulation in NPG mice. The sublethally irradiated NPG mice were transplanted with 4.0 × 104 CB 17/OE CD34+ cells, CTRL, or CB 17/KD CD34+ cells using intravenous injection. We temporally monitored the PB of NPG recipients transplanted with miR-17-modulated CB CD34+ cells for 20 weeks by analyzing the percentage of human CD45+ cells using flow cytometry every 4 weeks. Although the percentage of human CD45+ cells gradually increased in the PB from all of the mice transplanted with miR-17-modulated CB CD34+ cells, the PB from 17/KD recipients displayed a significantly higher percentage of human CD45+ cells at 4 weeks than CTRL recipients, indicating that the hematopoietic reconstitution potential of 17/KD CD34+ cells is higher than that of CTRL CD34+ cells during the first 4 weeks (Fig. 3a left panel). The percentage of human CD45+ cells in the PB from the 17/OE or 17/KD group showed a tendency, although insignificant, to be lower than that from the CTRL group at 20 weeks after transplantation. The levels of total human cell engraftment composed of CD45+, CD45−CD36+ and CD36−GPA+ cells, and CD45+CD34+ populations were assessed in the bone marrow MNCs of the engrafted mice at 20 weeks post-transplant using flow cytometry. At 20 weeks, the amount of total human cells in the mice injected with 17/OE CD34+ cells or 17/KD CD34+ cells was significantly lower than that of the mice injected with CTRL CD34+ cells (Fig. 3a middle panel; p < 0.03 and 0.04, respectively), which indicates that the hematopoietic reconstitution ability of 17/OE CD34+ cells and 17/KD CD34+ cells is reduced in comparison with that of CTRL CD34+ cells. The statistical analysis was performed by comparing the mice injected with 17/OE or 17/KD CD34+ cells to those injected with CTRL cells. We further analyzed the multilineage development from the input CB CD34+ cells. Flow cytometry analysis of the human graft in a representative engrafted mouse from each group is shown in Fig. 3b. There was no significant difference in the percentage of erythroid cells, including CD45−CD36+ and CD36−GPA+ populations upon comparing the 17/OE or 17/KD group with the corresponding CTRL group (Fig. 3a right panel). However, we observed a significantly lower percentage of CD45+ cells in the 17/OE group compared to that in the CTRL group. Similarly, in contrast to the CTRL group, the 17/OE group also showed a significantly lower percentage of CD45+CD34+ cells (Fig. 3a right panel). The percentage of CD45+CD34+ cells in the 17/KD group whose transplants of CD34+ cells with miR-17 knockdown showed a tendency, although insignificant, to be lower than that of mice receiving transplants of CTRL CD34+ cells. The observed multilineage development from input 17/KD CD34+ populations partly coincides with the results of expansion and colony forming assays in vitro. However, when the input cell numbers were similar, the 17/OE CD34+ cells contributed significantly less to hematopoietic reconstitution in recipient mice as opposed to the CTRL CD34+ cells, which was different from the in vitro expansion and colony forming assay, suggesting that the hematopoietic reconstitution capability of miR-17-overexpressed CB CD34+ cells were reduced in vivo. To confirm whether this inconsistency resulted from the change of miR-17 expression in vivo, we checked the levels of miR-17 expression in GFP-positive cells from engrafted mice at 20 weeks. As shown in Fig. 3c, the expression levels of miR-17 were still up- or downmodulated in GFP-positive cells from NPG recipients transplanted with CB 17/OE CD34+ cells or 17/KD CD34+ cells, respectively, although the expression levels of miR-17 became somewhat lower compared to that of corresponding initial cells. To further confirm that the human cells determined by flow cytometry were of human origin, the human-specific 17α-satellite gene was detected using PCR in several representative engrafted mice, which contained different percentages of human cells. We found that the human 17α-satellite gene could be detected by PCR amplification when the percentage of human cells was over 0.50 % (Fig. 3d, lanes 3–5), whereas it was undetectable at a percentage of 0.21 % (Fig. 3d, lane 2).Fig. 3


miR-17 promotes expansion and adhesion of human cord blood CD34(+) cells in vitro.

Yang Y, Wang S, Miao Z, Ma W, Zhang Y, Su L, Hu M, Zou J, Yin Y, Luo J - Stem Cell Res Ther (2015)

Effect of miR-17 modulation on the hematopoietic reconstitution potential of CB CD34+ cells in NPG mice. a Effect of miR-17 modulation on repopulation of CB CD34+ cells in NOD prkdcscid Il2rg (NPG™) mice. 4.0 × 104miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) CB CD34+ cells were injected intravenously into the sublethally irradiated NPG mice (n = 6 per group). The PB of NPG recipients was temporally monitored by analyzing CD45+ population every 4 weeks. The mice were sacrificed at 20 weeks after transplantation and the mononuclear cells from bone marrow were analyzed for human cells composed of CD45+, CD45−CD36+ and CD36−GPA+ cells and CD45+CD34+ population by flow cytometry. The level of total human cell engraftment is shown in the left panel. p < 0.03 or 0.04, between the NPG mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells (Student’s t-test). The fraction of CD45+, erythroid (CD45−CD36+ and CD36−GPA+) and CD45+CD34+ population among the engrafted human cells is shown in the middle panel. The percent of CD45+ cells of PB from NPG recipients was shown in the right panel. *p < 0.05, between the NPG mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells (Student’s t-test). The significant difference was analyzed between the mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells. b Flow cytometry analysis of the human CB CD34+ cell repopulation in a representative NPG mouse after miR-17 modulation. Fresh CD34+ cells served as controls. The MNCs from bone marrow harvested from the engrafted NPG mice were examined by flow cytometry for the assessment of human cells composed of CD45+ cells (R1), erythroid cells including CD45−CD36+ (R2) and CD36−GPA+ (R3) populations and CD45+CD34+ cells (R4). c The levels of miR-17 expression in GFP-positive cells from engrafted mice at 20 weeks were tested by real-time PCR. d The bone marrow MNCs containing the different percentage of human cells (lanes 2–5) from the representative engrafted mice were analyzed for human-specific 17α-satellite DNA by PCR. The human-specific 17α-satellite gene was detected when the human cells were over 0.50 % (lanes 3–5) whereas it was indetectable at a percentage of 0.21 % (lane 2). Lane 1, one mouse without transplants; lane 2, one mouse receiving transplants of 17/OE CD34+ cells; lane 3, one mouse receiving transplants of 17/KD CD34+ cells; lane 4, one mouse receiving transplants of CTRL CD34+ cells; lane 5, one mouse receiving transplants of fresh CD34+ cells
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Fig3: Effect of miR-17 modulation on the hematopoietic reconstitution potential of CB CD34+ cells in NPG mice. a Effect of miR-17 modulation on repopulation of CB CD34+ cells in NOD prkdcscid Il2rg (NPG™) mice. 4.0 × 104miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) CB CD34+ cells were injected intravenously into the sublethally irradiated NPG mice (n = 6 per group). The PB of NPG recipients was temporally monitored by analyzing CD45+ population every 4 weeks. The mice were sacrificed at 20 weeks after transplantation and the mononuclear cells from bone marrow were analyzed for human cells composed of CD45+, CD45−CD36+ and CD36−GPA+ cells and CD45+CD34+ population by flow cytometry. The level of total human cell engraftment is shown in the left panel. p < 0.03 or 0.04, between the NPG mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells (Student’s t-test). The fraction of CD45+, erythroid (CD45−CD36+ and CD36−GPA+) and CD45+CD34+ population among the engrafted human cells is shown in the middle panel. The percent of CD45+ cells of PB from NPG recipients was shown in the right panel. *p < 0.05, between the NPG mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells (Student’s t-test). The significant difference was analyzed between the mice injected with 17/OE or 17/KD CD34+ cells and those injected with CTRL cells. b Flow cytometry analysis of the human CB CD34+ cell repopulation in a representative NPG mouse after miR-17 modulation. Fresh CD34+ cells served as controls. The MNCs from bone marrow harvested from the engrafted NPG mice were examined by flow cytometry for the assessment of human cells composed of CD45+ cells (R1), erythroid cells including CD45−CD36+ (R2) and CD36−GPA+ (R3) populations and CD45+CD34+ cells (R4). c The levels of miR-17 expression in GFP-positive cells from engrafted mice at 20 weeks were tested by real-time PCR. d The bone marrow MNCs containing the different percentage of human cells (lanes 2–5) from the representative engrafted mice were analyzed for human-specific 17α-satellite DNA by PCR. The human-specific 17α-satellite gene was detected when the human cells were over 0.50 % (lanes 3–5) whereas it was indetectable at a percentage of 0.21 % (lane 2). Lane 1, one mouse without transplants; lane 2, one mouse receiving transplants of 17/OE CD34+ cells; lane 3, one mouse receiving transplants of 17/KD CD34+ cells; lane 4, one mouse receiving transplants of CTRL CD34+ cells; lane 5, one mouse receiving transplants of fresh CD34+ cells
Mentions: To further support our in vitro expansion results, we examined the hematopoietic reconstitution potential of CB CD34+ cells after miR-17 modulation in NPG mice. The sublethally irradiated NPG mice were transplanted with 4.0 × 104 CB 17/OE CD34+ cells, CTRL, or CB 17/KD CD34+ cells using intravenous injection. We temporally monitored the PB of NPG recipients transplanted with miR-17-modulated CB CD34+ cells for 20 weeks by analyzing the percentage of human CD45+ cells using flow cytometry every 4 weeks. Although the percentage of human CD45+ cells gradually increased in the PB from all of the mice transplanted with miR-17-modulated CB CD34+ cells, the PB from 17/KD recipients displayed a significantly higher percentage of human CD45+ cells at 4 weeks than CTRL recipients, indicating that the hematopoietic reconstitution potential of 17/KD CD34+ cells is higher than that of CTRL CD34+ cells during the first 4 weeks (Fig. 3a left panel). The percentage of human CD45+ cells in the PB from the 17/OE or 17/KD group showed a tendency, although insignificant, to be lower than that from the CTRL group at 20 weeks after transplantation. The levels of total human cell engraftment composed of CD45+, CD45−CD36+ and CD36−GPA+ cells, and CD45+CD34+ populations were assessed in the bone marrow MNCs of the engrafted mice at 20 weeks post-transplant using flow cytometry. At 20 weeks, the amount of total human cells in the mice injected with 17/OE CD34+ cells or 17/KD CD34+ cells was significantly lower than that of the mice injected with CTRL CD34+ cells (Fig. 3a middle panel; p < 0.03 and 0.04, respectively), which indicates that the hematopoietic reconstitution ability of 17/OE CD34+ cells and 17/KD CD34+ cells is reduced in comparison with that of CTRL CD34+ cells. The statistical analysis was performed by comparing the mice injected with 17/OE or 17/KD CD34+ cells to those injected with CTRL cells. We further analyzed the multilineage development from the input CB CD34+ cells. Flow cytometry analysis of the human graft in a representative engrafted mouse from each group is shown in Fig. 3b. There was no significant difference in the percentage of erythroid cells, including CD45−CD36+ and CD36−GPA+ populations upon comparing the 17/OE or 17/KD group with the corresponding CTRL group (Fig. 3a right panel). However, we observed a significantly lower percentage of CD45+ cells in the 17/OE group compared to that in the CTRL group. Similarly, in contrast to the CTRL group, the 17/OE group also showed a significantly lower percentage of CD45+CD34+ cells (Fig. 3a right panel). The percentage of CD45+CD34+ cells in the 17/KD group whose transplants of CD34+ cells with miR-17 knockdown showed a tendency, although insignificant, to be lower than that of mice receiving transplants of CTRL CD34+ cells. The observed multilineage development from input 17/KD CD34+ populations partly coincides with the results of expansion and colony forming assays in vitro. However, when the input cell numbers were similar, the 17/OE CD34+ cells contributed significantly less to hematopoietic reconstitution in recipient mice as opposed to the CTRL CD34+ cells, which was different from the in vitro expansion and colony forming assay, suggesting that the hematopoietic reconstitution capability of miR-17-overexpressed CB CD34+ cells were reduced in vivo. To confirm whether this inconsistency resulted from the change of miR-17 expression in vivo, we checked the levels of miR-17 expression in GFP-positive cells from engrafted mice at 20 weeks. As shown in Fig. 3c, the expression levels of miR-17 were still up- or downmodulated in GFP-positive cells from NPG recipients transplanted with CB 17/OE CD34+ cells or 17/KD CD34+ cells, respectively, although the expression levels of miR-17 became somewhat lower compared to that of corresponding initial cells. To further confirm that the human cells determined by flow cytometry were of human origin, the human-specific 17α-satellite gene was detected using PCR in several representative engrafted mice, which contained different percentages of human cells. We found that the human 17α-satellite gene could be detected by PCR amplification when the percentage of human cells was over 0.50 % (Fig. 3d, lanes 3–5), whereas it was undetectable at a percentage of 0.21 % (Fig. 3d, lane 2).Fig. 3

Bottom Line: However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells.The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical & Research Technology, School of Medicine, University of Maryland, Baltimore, MD, 21201, USA. yangyx@bjmu.edu.cn.

ABSTRACT

Introduction: We have recently found that miR-17 is necessary in the cell-extrinsic control of cord blood (CB) CD34(+) cell function. Here, we demonstrated that the proper level of miR-17 is also necessary in the cell-intrinsic control of the hematopoietic properties of CB CD34(+) cells.

Methods: The miR-17 overexpression and knockdown models were created using primary CB CD34(+) cells transfected by the indicated vectors. Long-term culture, colony forming, adhesion and trans-well migration assays were carried out to investigate the function of miR-17 on CB CD34(+) cells in vitro. NOD prkdc (scid) Il2rg () mice were used in a SCID repopulating cell assay to investigate the function of miR-17 on CB CD34(+) cells in vivo. A two-tailed Student's t-test was used for statistical comparisons.

Results: In vitro assays revealed that ectopic expression of miR-17 promoted long-term expansion, especially in the colony-forming of CB CD34(+) cells and CD34(+)CD38(-) cells. Conversely, downregulation of miR-17 inhibited the expansion of CB CD34(+) cells. However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells. The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.

Conclusion: We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

No MeSH data available.


Related in: MedlinePlus